achieved by treating the synthesized 2,2-dimethyl-3-oxooctanoyl-succinimide ester with free coenzyme A (CoA) acid under nitrogen environment (Scheme 18). The free CoA acid was deprotonated under basic condition and readily attacked the carbonyl carbon of the ester starting material, displacing the succinimide group. The crude product was extracted and ran on preparative HPLC to collect the desired pure 2,2-dimethyl-3- oxooctanoyl-CoA product. In HPLC chromatogram shown in Figure 29, 2,2-dimethyl- 3-oxooctanoyl-CoA eluted at 11.1 minutes. Mass spectral data using ESI-TOF for 2,2- dimethyl-3-oxooctanoyl-CoA molecule was also obtained to confirm its identity (Figure B22).
Figure 29. HPLC chromatogram of 2,2-dimethyl-3-oxoC8-CoA molecule using preparative HPLC. Excess of 2,2-dimethyl-3-oxoC8-succinimide ester and unreacted
free CoA eluted from 2.5 – 4.8 minutes. Only peak corresponding to 2,2-dimethyl-3- oxoC8-CoA product of interest was collected by fraction collector. 2,2-Dimethyl-3- oxooctanoyl-CoA eluted at 11.1 – 12.0 minutes.
Syntheses of 2,2-dimethyl-3-oxoC8-ACP and C10-ACP proteins
Enzymatic syntheses of E. coli 2,2-dimethyl-3-oxoC8-ACP and C10-ACP proteins were in a similar fashion as discussed in acyl-ACP protein syntheses for EsaI (chapter 2, Scheme 12). The syntheses of all acyl-ACPs were confirmed by HPLC and ESI-TOF (Figures 30 and B37).
The completion of the reactions was monitored by the depletion of apo-ACP at 7.5 minutes and the growth of corresponding acyl-ACP peak. The Sfp enzyme eluted at 5.1 minutes. Once the acyl-ACP reaction had gone to completion, Sfp enzyme was precipitated out by addition of ammonium sulfate to 75 % saturation and removed from the reaction mixture by centrifugation.58 Clean acyl-ACP product from the reaction was obtained by multiple washes with nanopure water to remove excess acyl-CoA and ammonium sulfate. These impurities absorbed at 260 nm while apo-ACP and acyl-ACP proteins absorbed at 280 nm on UV-Vis. The reduction of 260 peak after washes indicated the acyl-ACP protein was free of contamination.
Figure 30. HPLC chromatograms of apo-ACP and alternative 3-oxoacyl-ACPs using analytical HPLC over a period of 10 minutes with flow rate of 600 L/min.
In the YspI-catalyzed reaction, the formation of MTA and holo-ACP products after quenching the enzyme were measured quantitatively by using the same HPLC methods discussed in chapter 2 used for determining rates of EsaI. Product formation over a 6 minutes period was linear, indicating that 4 minutes of incubation with enzyme would be sufficient to determine the initial reaction rate (Figure 31).
Figure 31. Different quenched time (0, 2, 4, 6 minutes) follwing formation of MTA and holo-ACP in lactonization and acylation half-reactions in YspI. In the
reaction, concentrations of YspI, SAM, and 2-furoyl-ACP were fixed at 1.34 M, 500 M, and 112 M, respectively. In lactonization, MTA peak at 0 minute corresponded to contaminant MTA in commercially available SAM from Sigma. In acylation, decrease in furoyl-ACP peak area was accompanied by increase in holo-ACP product peak area.
In the lactonization half-reaction, SAM and MTA peaks eluted at 0.65 and 5.05 minutes, respectively (Figure 32). Since MTA is a common contaminant in commercial SAM, the actual amount of MTA produced in the quenched reaction was determined by subtracting the total peak area from the contaminant MTA peak area presented in the background. The MTA concentration produced in the enzymatic reaction was calculated
from its respective peak area by using MTA standard curve. In acylation half-reaction, holo-ACP and acyl-ACP (2,2΄-dimethyl-3-oxoC8-ACP in this case) eluted at 6.8 and 8.0 minutes, respectively (Figure 33). The overlaid background and YspI-catalyzed reaction chromatograms showed that in the presence of YspI, holo-ACP peak increased while 2,2΄-dimethyl-3-oxoC8-ACP decreased relatively.
Figure 32. Lactonization assay for YspI. HPLC chromatogram of MTA product
formation in YspI-catalyzed reaction using 0 M, 10 M, and 20 M 2,2΄-dimethyl-3- oxoC8-ACP substrate concentrations, fixed 500 µM SAM, fixed 1 µM YspI quenched at 4 minutes with 6 M HCl. SAM eluted at 0.65 minutes; MTA peak from SAM lactonization eluted at 5.05 minutes. The peak areas were reported in arbitrary units. The actual amount of MTA produced in the quenched reaction was determined by subtracting the total peak area from the contaminant MTA peak area presented in the background. The MTA concentration produced in the enzymatic reaction was calculated from its respective peak area by using MTA standard curve.
Figure 33. Acylation assay for YspI. HPLC chromatogram of holo-ACP product
formation in YspI-catalyzed reaction using 70 M (in blue) and 20 M (in pink) 2,2΄- dimethyl-3-oxoC8-ACP substrate concentration, fixed 500 µM SAM, fixed 1 µM YspI quenched at 4 minutes with 4 M acetate buffer, pH 3.6. Black chromatogram is background with 2,2΄-dimethyl-3-oxoC8-ACP eluted at 8.0 minutes. Blue and pink chromatograms are YspI-catalyzed reactions with the appearance of holo-ACP peak at 6.8 minutes. An increase in holo-ACP peak was accompanied by a decrease in 2,2΄- dimethyl-3-oxoC8-ACP peak area in YspI-catalyzed reaction. The peak areas were reported in arbitrary units. The holo-ACP concentration in the quenched reaction mixture was determined from its respective peak area by using holo-ACP standard curve.
Determination of kinetic parameters of alternative 3-oxoacyl-ACP substrates with YspI wildtype
Previously, mass spectrometry study had suggested that YspI produced majority 3-oxoC8-HSL, but the importance of the oxygen and hybridization at C-3 position had not yet been elucidated.66 In this thesis, we were interested in exploring the effect of change of heteroatom (oxygen to sulfur and nitrogen), methylation of 3-oxoC8-ACP substrate, change of hybridization at C-3, change of straight acyl-chain to aromatic analogs, change of position of oxygen atom in the acyl-chain, change of acyl-chain length, and removal of oxygen atom in the acyl-chain on YspI activity.
The initial rate at each substrate concentration was fitted to Michaelis-Menten equation to determine kinetic constants (Figure C4 and C5). The Km, kcat, and kcat/Km
values in lactonization and acylation assay were within error (Table 4). A general Km trend was observed among all alternative substrates: the substrates that carried oxygen at C-3 position had similar range of Km value; as the oxygen atom moved to different position (C-2, C-4, and C-5) or removed, or acyl chain length changed from 8 carbons to 6 carbons, Km increased and kcat decreased. This variation in activity can be truly
understood when comparing catalytic efficiencies (kcat/Km) of alternative substrates all together. The relative catalytic efficiencies of all substrates were compared in Figure 34.
100 (M) (min-1) (M-1min-1) 2-Benzofuranacetyl-ACP (13) 7.71 ± 1.19 3.28 ± 0.12 0.425 ± 0.068 100.00 2,2-Dimethyl-3-oxoC8-ACP (14) 7.78 ± 0.89 1.85 ± 0.05 0.238 ± 0.028 56.03 2-Furanacetyl-ACP (1) 8.35 ± 1.64 0.701 ± 0.032 0.0840 ± 0.0169 19.76 2-Tetrahydrofuranacetyl-ACP (2) 8.19 ± 1.46 0.610 ± 0.029 0.0744 ± 0.0137 17.52 2-Pyridylacetyl-ACP (4) 10.21 ± 1.49 0.742 ± 0.028 0.0727 ± 0.0110 17.12 2-Furoyl-ACP (5) 15.79 ± 3.12 0.574 ± 0.035 0.0364 ± 0.0075 8.56 5-oxoC8-ACP (11) 23.42 ± 3.98 0.788 ± 0.041 0.0336 ± 0.0060 7.92 4-oxoC8-ACP (10) 37.37 ± 5.19 1.12 ± 0.051 0.0299 ± 0.0044 7.06 5-oxoC6-ACP (7) 25.71 ± 2.44 0.619 ± 0.017 0.0241 ± 0.0024 5.67 4-oxoC6-ACP (6) 32.34 ± 4.66 0.593 ± 0.026 0.0183 ± 0.0028 4.32 C8-ACP (12) 87.87 ± 16.83 1.22 ± 0.093 0.0138 ± 0.0028 3.26 C6-ACP (9) 97.72 ± 15.90 0.780 ± 0.055 0.0080 ± 0.0014 1.88 C10-ACP (15) 112.60 ± 16.86 0.562 ± 0.038 0.0050 ± 0.0008 1.18 2-Thiopheneacetyl-ACP (3) 2.81 ± 0.89 0.774 ± 0.115 0.275 ± 0.097 64.84 a % activity = 𝑘𝑐𝑎𝑡 𝐾𝑚 ⁄ 0.425 × 100
101 (B) Acylation Km (M) kcat (min-1) kcat/Km (M-1min-1) % activity a 2-Benzofuranacetyl-ACP (13) 7.68 ± 1.11 3.24 ± 0.12 0.422 ± 0.063 100.00 2,2-Dimethyl-3-oxoC8-ACP (14) 7.09 ± 0.64 1.79 ± 0.04 0.253 ± 0.023 59.91 2-Furanacetyl-ACP (1) 8.86 ± 0.85 0.864 ± 0.020 0.0975 ± 0.0096 23.12 2-Tetrahydrofuranacetyl-ACP (2) 7.49 ± 0.93 0.643 ± 0.019 0.0858 ± 0.0110 20.34 2-Pyridylacetyl-ACP (4) 9.64 ± 0.95 0.768 ± 0.0206 0.0797 ± 0.0081 18.88 5-oxoC8-ACP (11) 21.79 ± 2.80 1.066 ± 0.03917 0.0489 ± 0.0066 11.59 2-Furoyl-ACP (5) 15.90 ± 2.78 0.707 ± 0.036 0.0445 ± 0.0081 10.54 4-oxoC8-ACP (10) 36.56 ± 5.25 1.29 ± 0.061 0.0353 ± 0.0053 8.37 5-oxoC6-ACP (7) 28.05 ± 3.49 0.704 ± 0.027 0.0251 ± 0.00327 5.95 4-oxoC6-ACP (6) 34.22 ± 3.91 0.649 ± 0.025 0.0190 ± 0.0023 4.50 C8-ACP (12) 83.89 ± 16.08 1.34 ± 0.098 0.0159 ± 0.0033 3.78 C6-ACP (9) 97.73 ± 12.07 0.841 ± 0.038 0.0086 ± 0.0011 2.04 C10-ACP (15) 110.50 ± 10.08 0.579 ± 0.018 0.0052 ± 0.0005 1.24 2-Thiopheneacetyl-ACP (3) 1.94 ± 0.51 0.659 ± 0.067 0.339 ± 0.095 80.31 a % activity = 𝑘𝑐𝑎𝑡 𝐾𝑚 ⁄ 0.422 × 100
Table 5. Effect of 2-thiopheneacetyl-ACP on YspI activity when SAM is fixed.
Fixed S Variable S Km (M) kcat (min-1) kcat/Km (M-1min-1) Ki (M) 50 M SAM 2-Thiopheneacetyl-ACP 4.58 2.23 0.48 ± 0.12 0.104 ± 0.058 26.54 12.56 500 M SAM 2-Thiopheneacetyl-ACP 2.81 ± 0.89 0.774 ± 0.115 0.275 ± 0.097 30.31 9.18
Figure 34. Comparison of catalytic efficiency of the alternative 3-oxoacyl-ACP substrates for YspI by following Lactonization (A) and Acylation assay (B). 2-
Benzofuranacetyl-ACP with sp2 hybridized carbon at C-3 and eight carbons in acyl-chain length showed the highest catalytic efficiency among all of the tested substrates with YspI. The catalytic activity of 2-benzofuranacetyl-ACP was about 4-fold higher than that of 2- furanacetyl-ACP. The change of heteroatom, position of oxygen atom, removal of oxygen atom, and variation in acyl-chain length significantly affected the activity with YspI.
Among all of the substrates, 2-benzofuranacetyl-ACP showed the highest catalytic efficiency (0.425 ± 0.068 M-1min-1). Comparing kcat/Km values of all alternative 3- oxoacyl-ACP substrates, 2-benzofuranacetyl-ACP substrate had the catalytic efficiency within an order of magnitude to that observed for ACP-dependent AHL synthases, such as RhlI (0.3 0.1 M-1min-1), BjaI (0.5 0.2 M-1min-1), and BmaI1 (0.31 0.05 M-1min- 1). The second-best substrate with YspI was 2,2-dimethyl-3-oxoC8-ACP with k
cat/Km value of 0.238 ± 0.028 M-1min-1. The 2-benzofuranacetyl-ACP and 2,2-dimethyl-3- oxoC8-ACP substrates meet the three criteria suggested for YspI activity: 8 carbons in acyl chain, sp2 hybridized C-3, and oxygen at C-3. However, the catalytic efficiency of 2,2- dimethyl-3-oxoC8-ACP was 2-fold less active than 2-benzofuranacetyl-ACP. Similar to the situation of 2,2-dimethyl-3-oxoC6-ACP in EsaI, the two methyl groups at C-2 of 2,2-
dimethyl-3-oxoC8-ACP may possibly introduce some steric hindrance in acyl-chain binding site of YspI, thus slightly reducing the kcat and kcat/Km values.
In the YspI-catalyzed reaction, 2-furanacetyl-ACP had only about 19-23 % activity in compared to the EsaI-catalyzed reaction. Yet, 2-furanacetyl-ACP is very similar to 2- benzofuranacetyl-ACP in term of hybridization of C-3 and oxygen at C-3, except that 2- furanacetyl-ACP is smaller than benzofuranacetyl-ACP in molecular size. The catalytic efficiency of 2-furanacetyl-ACP decreased by 4-fold compared to 2-benzofuranacetyl- ACP in YspI because YspI has a deeper acyl-chain binding pocket compared to EsaI that could result in too much fluctuations of acyl chain in the acyl-chain binding pocket of YspI and make it difficult to dock the 2-furanacetyl-ACP substrate in a productive conformation. 2-Tetrahydrofuranacetyl-ACP substrate with sp3 hybridized C-3 instead of sp2 had a 5-fold decrease in catalytic efficiency compared to 2-benzofuranacetyl-ACP. There is not much different in catalytic efficiency between 2-tetrahydrofuranacetyl-ACP and 2-furanacetyl- ACP although the hybridization of carbon at C3 position is different.
The alteration of heteroatom from oxygen to nitrogen at C-3 position also affected the kcat/Km value drastically. The catalytic efficiency of 2-pyridylacetyl-ACP with nitrogen in place of oxygen at C-3 decreased by 6-fold compared to 2-benzofuranacetyl-ACP and 1-fold compared to 2-furanacetyl-ACP. Although the lone pair of electrons of nitrogen center of pyridine is not part of the -conjugated system and can serve as hydrogen bond acceptor/donor, kcat/Km value showed that 2-pyridylacetyl-ACP is less favorable than 2- benzofuranacetyl-ACP when bound to YspI.
Unlike EsaI, YspI shows a very distinctive loss in activity when position of oxygen in acyl-chain changed to C-2, C-4, and C-5. 2-Furoyl-ACP, 5-oxoC8-ACP, and 4-oxoC8-
Interestingly, when chain length of the substrate was shortened by 2 carbons with the same position of oxygen (5-oxoC6-ACP and 4-oxoC6-ACP), the catalytic efficiencies dropped by 2-fold compared to those of 5-oxoC8-ACP and 4-oxoC8-ACP. In YspI-catalyzed reaction, the kcat/ Km value of 5-oxoC8-ACP was about 1.4-fold higher than that of 5- oxoC6-ACP. Likewise, the kcat/Km value of 4-oxoC8-ACP was about 1.6-fold higher than that of 4-oxoC6-ACP. The data may suggest that acyl-chain of 8 carbons is able to reach to the bottom of the acyl-chain binding pocket more easily and produce easier productive conformation. However, the shorter acyl-chain in 4-oxoC6-ACP and 5-oxoC6-ACP may increase the flexibility of acyl-chain in the shorter substrates resulting in a nonproductive binding for the enzyme-substrate ternary complex. This will decrease the turnover number (kcat) of the substrate.
Furthermore, without a heteroatom in the acyl-chain, the activity of C6-ACP, C8- ACP, and C10-ACP dropped significantly (1 – 4 %) compared to the highest activity substrate, 2-benzofuranacetyl-ACP. The data indicated that the presence of a heteroatom plays a role in retaining the activity of YspI. In addition, the preference of YspI for a specific chain length was also examined by looking catalytic efficiencies of C6-ACP, C8- ACP, and C10-ACP. Although these three substrates do not carry any heteroatom in acyl- chain, but the difference in chain length may provide useful information about acyl-chain length specificity of YspI for acyl-ACP substrate. The catalytic efficiencies of these three substrates can be ranked in high to low order: C8-ACP > C6-ACP > C10-ACP. The activity
of C10-ACP was only 1 % compared to 2-benzofuranacetyl-ACP and 3-fold less than C8- ACP. This may be explained that the difference by 2 carbons in C10-ACP compared to C8- ACP may cause the acyl-chain to protrude from the V-cleft binding pocket in YspI. Looking at all 14 substrates together, it is clear that YspI exhibits a strong preference for substrates with length of 8 carbons.
Last but not least, 2-thiopheneacetyl-ACP is an interesting substrate. It exhibited substrate inhibition when SAM was fixed and 2-thiopheneacetyl-ACP was varied (Figure C4, C5, and C8). Table 5 summarizes kinetic parameters when SAM concentrations were fixed at 50 M and 500 M. The inhibition constant Ki value when fixed SAM at 500 M (30.31 9.18 M) was compared to Ki value when fixed SAM at 50 M (26.54 12.56 M). Substrate inhibition was observed in both of the cases and had similar Ki values.
Yet, when 2-thiopheneacetyl-ACP was fixed and SAM was the variable substrate, substrate inhibition was not observed (Figure C9). In addition, Km of SAM when fixed 2- benzofuranacetyl-ACP (75.98 17.49 M) was determined and compared to Km of SAM when fixed 2-thiopheneacetyl-ACP (23.52 4.03 M) (Figure C9). The kcat for fixed
concentrations of 2-thiopheneacetyl-ACP with variable concentration of SAM (0.764 0.029 min-1) is about 5-fold lower than that for fixed concentrations of 2-benzofuranacetyl- ACP (3.21 0.18 min-1). However, the k
cat/Km for fixed 2-thiopheneacetyl-ACP (0.0325 0.0057 M-1min-1) is almost similar to that for fixed 2-benzofuranacetyl-ACP (0.0423 0.0100 M-1min-1). Both 2-thiopheneacetyl-ACP and 2-benzofuranacetyl-ACP showed normal hyperbolic curves when SAM was the variable substrate. This result together with substrate inhibition data when SAM is fixed indicated that all available free enzyme (E) favored the formation of stable EA (E.2-thiopheneacetyl-ACP or E.2-benzofuranacetyl-
thiopheneacetyl-ACP in YspI can be described by random sequential mechanism discussed in chapter 2 (Figure 25). At low concentrations of 2-thiopheneacetyl-ACP, it populated the productive pathway by formation of E.2-thiopheneacetyl-ACP.SAM complex. However, when 2-thiopheneacetyl-ACP reached beyond saturation, the excess of 2-thiopheneacetyl- ACP could also bind to E.SAM complex, pushing the nonproductive pathway to compete with productive pathway. The result is a lower turnover number and the reaction rate decreased. Another explanation is that the binding of 2-thiopheneacetyl-ACP in the acyl- chain binding pocket of YspI partially locks the enzyme in a conformation that the substrate can’t be released to perform catalysis, resulting in decrease of released product.
As a result, these data indicated that the presence of heteroatom is important in maintaining activity of YspI, but chain length can also play a critical point in establishing the necessary interactions to position the acyl-chain into a productive conformation to undergo catalysis.
CHAPTER FOUR: CONCLUSIONS
In Gram-negative bacteria, AHL synthases catalyze the synthesis of AHL signal molecules using acyl-ACP and SAM substrates. Many therapeutically relevant AHL synthases utilize -ketoacyl-ACPs; yet, these enzymes remain uncharacterized with their native substrate due to the instability of the β-ketoacyl-ACP substrate in vitro. This thesis work is the first systematic investigation to design and evaluate the alternative 3-oxo-acyl- ACP substrates for Pantoea stewartii EsaI and Yersinia pestis YspI. This study will open new doors to explore inhibitors for several uncharacterized AHL synthase enzymes as well as other -ketoacyl-ACP utilizing enzymes that impacts human health.
From the kinetic study of EsaI T140A mutant with C6-ACP, it was confirmed that the acyl-chain binding pocket of EsaI lost specificity with respect to the - position of the acyl chain when the key threonine residue was mutated to alanine (Figure 34). The kinetic studies of the alternative 3-oxoacyl-ACP substrate analogs with EsaI wild-type suggest that the presence of an oxygen at the -carbon position and the chain length of six carbons are preferable to yield high catalytic efficiency. When studying kinetics of the alternative 3- oxoacyl-ACP substrate analogs with YspI wild-type, our data indicate that YspI shows a strong preference for substrate with an acyl-chain length of eight carbons and an oxygen heteroatom at the -carbon. From this thesis work, we found that 2-furanacetyl-ACP and 2-benzofuranacetyl-ACP are the best alternative substrate for EsaI and YspI, respectively. The catalytic efficiencies of these substrates are within an order of magnitude to that observed for ACP/CoA-dependent AHL synthases with their native acyl-ACP/ acyl-CoA
activity. Finally, the substrate with sulfur atom (2-thiophenacetyl-ACP) in place of oxygen at the -carbon exhibits substrate inhibition in both EsaI and YspI enzymes, suggesting a possibility of random sequential mechanism. Future product inhibition experiments are needed to confirm this observation.
Figure 35. Comparison catalytic efficiency analysis of characterized AHL synthases with their native substrate. 2-Furanacetyl-ACP and 2-benzofuranacetyl-ACP
are the best alternative substrate for EsaI and YspI, respectively. The catalytic efficiencies of these two substrates are within an order of magnitude to that observed for ACP/CoA- dependent AHL synthases with their native acyl-ACP/ acyl-CoA substrate, such as RhlI, BmaI, and BjaI.
In summary, this project met the criteria proposed in my thesis objective. The success of synthesis and high activity of β-ketoacyl-ACP mimics for EsaI and YspI should open new doors in characterizing this class of enzymes. Moreover, the β-ketoacyl-ACP substrates could be used as chemical probes to explore and design inhibitors for therapeutically important AHL synthases and several uncharacterized enzymes that impacts human health, such as -ketoacyl-ACP reductase in fatty acid biosynthesis, polyketide synthase in polyketide synthesis which are targets for antimicrobial, antimalarial, and anti-cancer drugs.
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